PROJECT SUMMARY Acute Myeloid Leukemia (AML) is the 2nd most common type of leukemia diagnosed with a 5-year survival rate of only 27%. Though dose-intensive induction and consolidation chemotherapy induces clinical complete remission in the majority of patients suitable for treatment, most relapse whereas others develop refractory AML. Recently, the Bcl-2 inhibitor, venetoclax, demonstrated improved clinical outcomes when combined with the conventional therapeutic, cytarabine (AraC), in relapsed and refractory AML. Based upon efficacy in preclinical models, we hypothesize that liposomal delivery of the pro-apoptotic bioactive lipid C6-ceramide will augment the efficacy of this low dose AraC/venetoclax standard-of-care regimen and have a meaningful clinical impact in treating relapsed/refractory AML. This hypothesis will be examined in three Specific Aims. In Specific Aim 1, a Phase 1b (dose escalation)/2a (dose-expansion) clinical trial for ceramide nanoLiposomes (CNL) in relapsed/refractory AML patients treated in combination with low dose AraC and venetoclax (pre-IND #142902, UVA Protocol Review Committee Approval #5414, CAV trial) will be conducted. The premise of this trial is supported by the fact that CNL has already reached its putative MTD in an FDA (IND 109471, NCT02834611) NCI-supported (U43 CA186118) Phase 1 trial for solid tumors, where CNL has been well- tolerated with multiple patients exhibiting stable disease. In Specific Aim 2, we examine the underlying cooperativity between CNL, AraC, and venetoclax in AML. We show that co-administration of CNL with AraC and/or venetoclax exerts multiple synergistic mechanisms of efficacy. First, we demonstrate that this regimen increases the ratio of pro-apoptotic C16- and C18-ceramides over less apoptotic C24 ceramides. Utilizing molecular strategies in both in vitro and in vivo models, we will test the hypothesis that this is mediated by ceramide synthases and determine how ceramide synthases regulate AML survival. Second, we show that CNL reduces venetoclax-induced elevation of pro-survival proteins. We will test the hypothesis that this effect is STAT3-dependent using molecular approaches in preclinical models. In Specific Aim 3, we show that the ratio of C18 to C24 ceramides predicts progression-free survival in AML patients, which is the first application of this ratio as a biomarker for cancer. We will extend and confirm sphingolipid metabolites or ratios as biomarkers of AML biology and prognosis utilizing large, well-characterized AML samples as a validation cohort. while also investigating clinical samples from Specific Aim 1 to assess biomarkers of CNL efficacy. Published and unpublished observations demonstrate that high-expression of enzymes that decrease ceramide levels (acid ceramidase, sphingosine kinase, glucosylceramide synthase) and reduce pro-apoptotic ceramides (e.g. C16/C18) leads to lower survival in AML patients. Together, the proposed studies will test the clinical application and unravel the mechanistic pathways of CNL cooperativity with AraC/venetoclax, while ascertaining the prognostic impact of dysregulated sphingolipid metabolism in AML.